The global objective of the proposed research is to biochemically characterize the chromatin-specific mechanisms by which members of the pRB family repress transcription, pRB has been shown in vitro and in vivo to function in the repression of many genes required for cell cycle progression. Indeed, mutations in the RB gene occur frequently in many kinds of cancer, implicating its role as a global regulator of cell division and differentiation. Available evidence suggests pRB functions by at least two distinct mechanisms. In the first, pRB binds to E2F and prevents the subsequent recruitment of TFIID and TFIIA, thus preventing formation of a pre-initiation complex (PIC). Recent work has shown that pRB functions by another E2F-independent mechanism dubbed "active" repression that appears to involve the repression of chromatin remodeling. To gain a better understanding of this mechanism the following aims will be undertaken: (1) analysis of chromatin-modifying and -remodeling mechanisms achieved through E2F, Sp 1, and pRB family members on synthetic promoters in vitro, (2) analysis of regulation of mouse E2fl promoter in vitro by E2F, Spl, and pRB family members, and (3) study contribution of mechanisms characterized in Aims 1 and 2 to mouse E2fl promoter in living cells. In vitro transcriptional analysis, Chromatin Immunoprecipitation (ChIP) analysis, and luminescence based transcriptional analysis will be the main technical methods utilized in these aims in order to bridge basic biochemical analysis of transcription with physiologically relevant mechanisms controlling cell cycle progression.